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Addressing protein synthesis regulation within small numbers of discrete neurons

解决少量离散神经元内的蛋白质合成调节问题

基本信息

批准号：
10091418
负责人：
MICHAEL ROSBASH
金额：
$ 32.72万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10091418
关键词：
Address Behavior Binding Biochemical Biological Biological Assay Biology Brain Catalytic Domain Cell physiology Cells Circadian Rhythms Clock protein Clustered Regularly Interspaced Short Palindromic Repeats Dimerization Drosophila genus Enzymes Event Fusion Protein Expression Goals Grant Health Human In Vitro Intention Knock-in Light Mammals Measurement Mediating Mental disorders Messenger RNA Methods Molecular Biology Motor Activity Nerve Degeneration Neurons Neurosciences Noise Orthologous Gene Paper Pharmaceutical Preparations Poly(A)-Binding Proteins Post-Transcriptional Regulation Protein Biosynthesis Proteins Publications RNA RNA Editing RNA Recognition Motif RNA Sequences RNA-Binding Proteins Regulation Ribosomal Proteins Ribosomes Signal Transduction Source Stroke Substance Addiction Substance abuse problem System Techniques Time Transcript Translating Translational Regulation Translations Tribes Variant Work Yeasts brain cell circadian fly improved in vivo interest novel novel strategies overexpression programs protein TDP-43 response tissue/cell culture transcriptome sequencing translation factor

项目摘要

Project Summary/Abstract Recent publications indicate that post-transcriptional regulation makes an important contribution to circadian rhythms, in flies and in mammals. This evidence is now quite extensive and includes translational regulation, which impacts the synthesis of key clock proteins within the mammalian SCN as well as within the small number of central brain neurons that govern Drosophila circadian locomotor activity rhythms. These neurons pose a significant challenge – and opportunity – for circadian biology and for molecular biology more generally: how can one assess biochemical events within a few neurons? For almost all measurements, there is not enough signal and/or a problematic signal:noise ratio when an extract is made and assayed starting with only a few cells/brain. Even methods that tag brain neurons are plagued with this problem if the fraction of tagged neurons/brain is very low. To address this issue in the context of RNA binding proteins and their targets, my lab developed a new technique, which we call TRIBE (Targets of RNA Binding proteins Identified by Editing). It fuses the catalytic domain of the RNA editing enzyme ADAR to RNA binding proteins. TRIBE takes advantage of the fact that one can make clean RNA and sequence it even from a single cell despite the inability to work with an extract from the same source material. We have used TRIBE with three different RBPs and also showed that it can work within the “small number of specific neurons” context. We have recently validated TRIBE and improved its efficacy, so that it has many fewer false negatives, i.e., it recognizes a much greater fraction of RBP- associated mRNAs. We will extend the method in two new translation-centric directions: to identify specific targets of the important translation factor eIF4E-BP and to identify ribosome-associated transcripts. We also propose to address some remaining issues, and extend TRIBE in new directions, with CRISPR-mediated knock-ins as well as with dimerization approaches. The latter will bring the editing moiety to the RNA on command, i.e., in response to a drug- or light-mediated dimerization signal. In all cases, assaying successfully small numbers of discrete neurons is the key biological focus. It is an important goal relevant to many human health problems like mental illness, neurodegeneration, stroke, substance abuse and addiction.

项目概要/摘要最近的出版物表明转录后调控对果蝇和哺乳动物的昼夜节律。这些证据现在相当广泛，包括翻译调节，影响哺乳动物 SCN 内关键时钟蛋白的合成以及控制果蝇昼夜运动的少数中枢神经元活动节奏。这些神经元对昼夜节律生物学和生物钟提出了重大挑战和机遇。对于更广泛的分子生物学来说：如何评估几个神经元内的生化事件？为了几乎所有测量都没有足够的信号和/或有问题的信噪比仅用几个细胞/大脑开始提取提取物并进行分析。甚至标记大脑神经元的方法也是如此如果标记的神经元/大脑的比例非常低，就会受到这个问题的困扰。为了解决这个问题在 RNA 结合蛋白及其靶标的背景下，我的实验室开发了一种新技术，我们称之为 TRIBE（通过编辑鉴定的 RNA 结合蛋白的靶标）。它融合了RNA的催化结构域将 ADAR 酶编辑为 RNA 结合蛋白。 TRIBE 利用了这样一个事实：人们可以制作尽管无法使用来自单个细胞的提取物，但仍可以清洁 RNA 并对其进行测序相同的来源材料。我们已经将 TRIBE 与三种不同的 RBP 一起使用，并且还表明它可以工作在“少量特定神经元”的背景下。我们最近验证了 TRIBE 并改进了它功效，因此它的假阴性要少得多，即它可以识别更大比例的 RBP- 相关 mRNA。我们将在两个新的以翻译为中心的方向上扩展该方法：重要翻译因子 eIF4E-BP 的特定靶点并鉴定核糖体相关成绩单。我们还建议解决一些遗留问题，并将 TRIBE 扩展到新的方向，通过 CRISPR 介导的基因敲入以及二聚化方法。后者将带来编辑根据命令，即响应药物或光介导的二聚化信号，将部分结合到RNA上。总共在这种情况下，成功检测少量离散神经元是关键的生物学焦点。它是一个与许多人类健康问题相关的重要目标，如精神疾病、神经退行性疾病、中风、药物滥用和成瘾。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Comparison of TRIBE and STAMP for identifying targets of RNA binding proteins in human and Drosophila cells.

DOI：
10.1261/rna.079608.123
发表时间：
2023-08
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
通讯作者：

TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins.

DOI：
10.1016/j.cell.2016.03.007
发表时间：
2016-04-21
期刊：
CELL
影响因子：
64.5
作者：
McMahon, Aoife C.;Rahman, Reazur;Jin, Hua;Shen, James L.;Fieldsend, Allegra;Luo, Weifei;Rosbash, Michael
通讯作者：
Rosbash, Michael

Protocol for using TRIBE to study RNA-protein interactions and nuclear organization in mammalian cells.